IDENTIFICATION OF GESTATIONALLY REGULATED GENES IN RAT MYOMETRIUM BY USE OF MESSENGER-RIBONUCLEIC-ACID DIFFERENTIAL DISPLAY

OBJECTIVE: We hypothesized that the proteins contributing to myometria l changes during gestation could be identified indirectly by analyzing the changing pattern of messenger ribonucleic acid expression in the myometrium during pregnancy, STUDY DESIGN: Ribonucleic acid was extrac ted from myometrium of timed pregnant Sprague-Dawley rats on days 12, 16, 20, 21, and 22 of pregnancy and on day 1 post partum. The techniqu e of messenger ribonucleic acid differential display, a simple and sen sitive polymerase chain reaction-based method for rapidly identifying messenger ribonucleic acids whose levels increase or decrease, was per formed with the nine different anchoring primers (oligodeoxythymidine( 11) VN: V = G, A, or C; N = G, A, or C) in combination with 24 differe nt 10-base oligonucleotides of random sequence. The polymerase chain r eaction products were separated by electrophoresis on a 5% polyacrylam ide sequencing gel, and those whose levels changed were then cloned, s equenced, and compared with those in the GenBank database to determine whether they corresponded to a known sequence in the database or were novel. Semiquantitative reverse transcriptase-polymerase chain reacti on was used to confirm differential expression of selected products. R ESULTS: Messenger ribonucleic acid differential display revealed >500 polymerase chain reaction products that were differentially expressed during gestation, 179 of which were cloned and sequenced. Of these, 15 7 were from messenger ribonucleic acids whose levels increased during gestation, and 22 were from transcripts that decreased. Eighty-seven ( 49%) were related to sequences in the GenBank database, of which 62 (3 5%) were from messenger ribonucleic acids encoding known proteins and 25 (14%) corresponded to known expressed sequence tags. The technique of semiquantitative reverse transcriptase-polymerase chain reaction co nfirmed the increased expression of messenger ribonucleic acids encodi ng beta-tropomyosin, type II phosphatidyl inositol-4-phosphate 5-kinas e, and a novel myometrial messenger ribonucleic acid named RPU0901AC. CONCLUSION: Messenger ribonucleic acid differential display is a simpl e and sensitive method for rapidly identifying myometrial messenger ri bonucleic acids that are differentially regulated during pregnancy. Th e identification of these differentially expressed messenger ribonucle ic acids may lead to a better understanding of the molecular basis of normal and abnormal parturition.